This application claims priority of International Application No. PCT/EP00/04493, filed May 18, 2000 and German Application No. 199 23 821.9, filed May 19, 1999, the complete disclosures of which are hereby incorporated by reference.
a) Field of the Invention
The invention is directed to a method and an arrangement for detecting the position of the scanned plane XY of an object and for its positioning in the focal plane Xxe2x80x2Yxe2x80x2 of a laser scanner, preferably in a laser scanning microscope.
b) Description of the Related Art
In the last two decades, scanners have achieved an advanced state of the art. Particularly in medical engineering, metrology and, above all, in microscopy, it is becoming more and more common for objects to be scanned by a laser beam. In the biological-medical field, for example, laser scanning microscopes have clear advantages over conventional microscopes which stem essentially from the higher resolution that can be achieved and the possibility of differentiating between determined depth layers.
Accordingly, in fluorescence microscopy, due to the high point intensity of the focused laser beam, it is possible to utilize the sensitivity of photomultipliers for image building when the fluorescence itself is weak. However, XY scanners require very high positioning accuracy in orientating the object to be scanned relative to the focusing plane of the microscope.
The precision with which the plane of the specimen to be analyzed must be brought into the focus of the laser beam in order to prevent intensity losses and avoid deficient image quality increases as depth resolution increases.
To this extent, in connection with the continuing development in this technical field, it is necessary to provide methods and arrangements by which the objects and planes to be scanned can be exactly and, if possible, automatically oriented relative to the focal plane. In general, requirements can no longer be met by the technique, still often applied, of positioning by means of sensitively adjustable manual drives with repeated position correction.
On this basis, it is the primary object of the invention to provide a method enabling an automatic and precise positioning of a scan plane XY in the focal plane Xxe2x80x2Yxe2x80x2 of a laser scanner.
According to the invention, it is provided in a method of the type described above that, after a rough orientation of the object carried out by fixing on an object holder, a laser beam is directed successively in time to at least three different points P1, P2 . . . Pn located in the scan plane XY of the object and, in doing so, each of the reflections proceeding from the points P1, P2 . . . Pn is imaged on a position-sensitive detector, an actual position value is determined at the detector for each reflection and is compared with a stored reference position value, actuating or adjustment commands for changing the inclination of the object holder are obtained from the deviations of the actual position values from the reference position values, and the inclination of the object holder is changed on the basis of these adjustment commands until points P1, P2 . . . Pn are located in the focal plane Xxe2x80x2Yxe2x80x2 of the laser scanner.
The essential advantages of this method consist in that, to a great extent, components which already exist in a laser scanner can be used for determining position, e.g., the position transmitter for the laser beam and the optical elements for transmitting the laser radiation reflected by the specimen to an optoelectronic reception device, followed by an evaluating unit. Further, positioning is not only more exact in this way, but is also substantially faster than in any of the previously known methods.
In a preferred embodiment of the method according to the invention, three points P1(x1;y1), P2(x2;y2) and P3(x3;y3), where y1=y2=x3=0 and x1=xe2x88x92x2xe2x89xa00; y3xe2x89xa00, are sampled, wherein one point P0, where x0=y0=0, lies approximately in the center of the object and/or of the plane XY to be scanned, P1, P2, P3 are preferably points near the edges of the object, and the deviations between the actual position values and reference position values are determined according to the principle of laser triangulation.
Accordingly, three points P1, P2, P3 are defined which are advantageously suited to position detection because, first, they are located far apart from each other due to their position at the edges of the object or at the edges of the plane XY to be scanned, which creates favorable preconditions for laser triangulation, and, second, they can be controlled in an uncomplicated manner assuming that point P0 approximately defines the starting position of the laser beam in the coordinate origin of the focal plane Xxe2x80x2Yxe2x80x2.
In triangulation, a light point is projected by means of a laser beam onto the plane of the measurement object to be scanned and the reflected light is imaged on a position-sensitive detector. The position of the reflection on the detector varies depending on the distance between the plane XY to be scanned and the objective, as measured in coordinate Z. The signal emitted by the detector is accordingly a measure for the distance between the reflecting surface and the objective or a measure of whether or not the reflecting point lies in the focus of the objective.
According to the invention, the object is initially oriented in such a way that the laser beam is directed, e.g., on position P0, the scanning device is then controlled in such a way that the laser beam is directed to point P1, a parallel displacement of the object holder in Z-direction is effected, if need be, until the reflection of P1 supplies a definite signal to the detector, the actual position value for point P1 is acquired, the scanning device is then controlled in such a way that the laser beam is directed to point P2, the actual position value for point P2 is now acquired, adjustment commands for tilting the object holder about the Y-axis are now determined from the deviations between the actual position values and reference position values, and the orientation of the X-axis of plane XY parallel to the focal plane Xxe2x80x2Yxe2x80x2 is brought about, the scanning device is then controlled in such a way that the laser beam is directed to point P3, the actual position value for point P3 is detected and, finally, an adjustment command for tilting the object holder about the X-axis is determined from the deviation of the actual position value from the reference position value of point P3 and, accordingly, the parallel orientation of the Y-axis of plane XY relative to the focal plane Xxe2x80x2Yxe2x80x2 is brought about.
In a particularly preferred construction of the invention, the steps indicated above are repeated in order to determine any existing deviations and to readjust in the described manner until no deviations are measurable and the planes XY are adjusted parallel with the focal plane Xxe2x80x2Yxe2x80x2.
Further, it lies within the framework of the invention that after this orientation of plane XY a parallel displacement in Z-direction is carried out, namely, until the reflections of P1, P2, P3 are imaged on the detector with maximum intensity. In this way, it is ensured with great dependability that the plane XY to be scanned is not only oriented parallel to the focal plane Xxe2x80x2Yxe2x80x2, but coincides with it.
It is a further object of the invention to provide an arrangement for carrying out the method steps mentioned above and for determining the position of the scan plane XY of an object and for its positioning in the focal plane Xxe2x80x2Yxe2x80x2 of a laser scanner, preferably a laser scanning microscope.
This object is met, according to the invention, by an arrangement which is outfitted with an object holder for receiving the object, with a photo-sensitive detector for locating the reflections of three points P1, P2, P3 which lie in plane XY and are sampled successively or simultaneously by a laser beam, with an evaluating circuit for determining deviations of the actual position value of each of these reflections from its reference position value on the detector, and with an adjusting device connected with the evaluating circuit for changing the inclination of the object holder relative to the focal plane Xxe2x80x2Yxe2x80x2 of the laser scanner.
The arrangement according to the invention is preferably directed to a laser scanner, known per se from the prior art, which has a mirror supported on a rotatable shaft for deflecting a laser beam in the direction of coordinate Xxe2x80x2 and in which the object holder is displaceable in a straight line in the direction of coordinate Yxe2x80x2, wherein the series of movements can be controlled and monitored by drives which are coupled with position transmitters.
In a preferred embodiment of the invention, the detector is constructed as a CCD line with 256 pixels and is a component part of a triangulation assembly in which there is further provided at least one laser diode as a separate radiation source for sampling points P1, P2, P3, preferably with a laser wavelength xcex greater than or equal to 780 nm and independent control, and optics for coupling the diode radiation, e.g., into the infinite beam path of the laser scanner.
It is particularly advantageous when the diode radiation has a Gaussian intensity profile and the actual positions of the diode beam reflected by points P1, P2, P3 on the CCD line are defined by the centroid of the Gaussian function. The steps for determining the centroid are known from the prior art and are therefore indicated only briefly herein.
First, the pixel with maximum intensity and the level of the base signal are determined on the CCD line. The half-height of the Gaussian function is calculated from these values and the intersections of the Gaussian function with the half-height are determined, wherein linear interpolation is carried out between the pixels. The mean value between the intersections approximates the centroid. By means of interpolation, the centroid is obtained with a resolution which is better than the pixel size and is only limited by the noise and deviations of the measurement signal from the assumed Gaussian function.
According to the invention, it is further provided that a storage is provided in the evaluating circuit with reference position values (associated with points P1, P2, P3), wherein the reference position is preferably defined in each instance in the center of the CCD line and, further, the evaluating unit has a subtracter, wherein an actual position value and a reference position value are applied to the inputs of this subtracter during every evaluation, with reference in each instance to one of the points P1, P2, P3, and a difference signal can be taken off at the output of the subtracter as an adjusting signal for the adjusting device coupled with the object holder.
The adjusting device is advantageously constructed in such a way that it has three drive elements which can be advanced separately in the direction of coordinate Z, each drive element being mechanically connected with the object holder via a separate articulation point A1, A2, A3. The articulation points A1, A2, A3 and points P1, P2, P3 lie in plane XY. Further, articulation points A1 and A2 are located along with points P1 and P2 on the same straight line which also passes through the coordinate origin P0 and accordingly forms the X-axis, while points A3, P3 and the coordinate origin likewise lie on a common straight line, namely, the Y-axis.
Due to this arrangement in connection with the separate controllability of the drive elements, it is ensured that the object holder can be tilted about the Y-axis, for example, when the articulation points A1 and A2 are controlled in opposite directions, while articulation point A3 remains stationary relatively. Further, this makes it possible to tilt the object holder in a defined manner about the X-axis insofar as only articulation point A3 is controlled and articulation points A1 and A2 remain stationary relatively.
This construction of the adjusting device according to the invention has the advantageous result that the angle adjusting movements about the X-axis on the one hand and Y-axis on the other hand can be carried out independent from one another and the X-axis need no longer be readjusted while the Y-axis is moved, and vice versa.
It is further advantageously provided that the drive elements are piezoelectric drives or, in an alternative construction, precision-movement threaded spindles coupled with rotating drives. Further, the adjusting mechanism can be constructed in such a way that the drive elements engage in recesses of the object holder, the drive elements contact the object holder within the recesses based on a ball-and-socket principle, and the drive elements and object holder are biased or pretensioned relative to one another elastically by means of springs so that they are mounted without play.
Of course, different constructions of the drive elements are possible: for example, pins which are displaceable in guides and which contact articulation points A1, A2, A3 on the one hand and are in contact with cam surfaces on the other hand, wherein the cams are driven by stepper motors which are outfitted with corresponding gear units for achieving the required precision.
For the sake of completeness, it is noted that while the invention has been explained with reference to the triangulation principle for detecting the laser radiation reflected by points P1, P2, P3, this does not rule out the use of other means for detecting the position of these reflections. In this respect, it is merely noted that it is possible to direct a collimated symmetric laser beam vertical to plane XY and to image the reflection on a quadrant diode via a cylindrical lens. In this regard, the ellipticity of the focus on the quadrant diode is a measure for the deviation of the position of the point acted upon by the laser beam from the focal plane. A contrast determining process can also be applied, for example, in which the distance between points P1, P2, P3 is changed during scanning until a contrast function derived from the determined image has reached its maximum. A simple contrast can be determined as well as the frequency spectrum of the image information.
The invention described above can be applied in a particularly advantageous manner for scanning the bottoms of microtiter plates and biochips containing fluorescing specimens. However, it must be taken into account in this regard that the laser radiation is reflected twice, namely, once by the underside and a second time by the inner side of the bottom of the plate. In other words, depending on spatial resolution, one or two laser reflections can appear on the CCD line. Since the inner side is closer to the specimen to be scanned, this reflecting surface is preferably used for orientation.
It may also be advantageous under certain circumstances when there is a second laser diode (in addition to the laser radiation source provided for fluorescence microscopy of the specimen) in the triangulation assembly instead of only one; although only one needs to be active at any time, the widely different apertures of different objectives can be taken into account in this way.
In another development, a mechanical shutter can be provided which blocks the microscope laser beam during the orientation of the plane XY. This prevents unwanted bleaching of the specimen.
The invention will be explained more fully in the following with reference to an embodiment example.